Plant oil is an economically important product not only due to its broad utilization in the food industry and as a component of feed ingredients but it also has a wide range of applications as biofuels or in the manufacture of various nutraceutical and industrial products. Within the plant itself, oil is essential to carry out a number of metabolic processes which are vital to growth and development particularly during seed germination and early plant growth stages. Considering its value, there is a growing research interest within the biotechnology field to improve plant oil production and make the supply more sustainable.
The major component of plant oil is triacylglyceride (TAG). It is the main form of storage lipid in oil seeds and the primary source of energy for seed germination and seedling development. TAG biosynthesis via the Kennedy pathway involves sequential acylation steps starting from the precursor sn-glycerol-3-phosphate (G3P). Firstly, G3P is esterified by an acyl-CoA to form lysophosphatidic acid (LPA) in a reaction catalyzed by glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15). This is followed by a second acylation step catalyzed by lysophosphatidic acid acyltransferase (LPAT; EC 2.3.1.51) forming phosphatidic acid (PA), a key intermediate in the biosynthesis of glycerolipids. The PA is then dephosphorylated by the enzyme phosphatidic acid phosphatase (PAP; EC3.1.3.4) to release the immediate precursor for TAG, the sn-1,2-diacylglycerol (DAG). Finally, DAG is acylated in the sn-3 position by the enzyme diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) to form TAG.
Since this last catalytic action is the only unique step in TAG biosynthesis, DGAT is termed as the committed triacylglycerol-forming enzyme. As DAG is located at the branch point between TAG and membrane phospholipid biosyntheses, DGAT potentially plays a decisive role in regulating the formation of TAG in the glycerolipid synthesis pathway (Lung and Weselake, 2006, Lipids. December 2006; 41(12):1073-88). There are two different families of DGAT proteins. The first family of DGAT proteins (“DGAT1”) is related to the acyl-coenzyme A:cholesterol acyltransferase (“ACAT”) and has been described in the U.S. Pat. Nos. 6,100,077 and 6,344,548. A second family of DGAT proteins (“DGAT2”) is unrelated to the DGAT1 family and is described in PCT Patention Publication WO 2004/011671 published Feb. 5, 2004. Other references to DGAT genes and their use in plants include PCT Publication Nos. WO2004/011,671, WO1998/055,631, and WO2000/001,713, and US Patent Publication No. 20030115632.
DGAT1 is typically the major TAG synthesising enzyme in both the seed and senescing leaf (Kaup et al., 2002, Plant Physiol. 129(4):1616-26; for reviews see Lung and Weselake 2006, Lipids. 41(12):1073-88; Cahoon et al., 2007, Current Opinion in Plant Biology. 10:236-244; and Li et al., 2010, Lipids. 45:145-157).
Raising the yield of oilseed crops (canola, sunflower, safflower, soybean, corn, cotton, linseed, flax etc) has been a major target for the agricultural industry for decades. Many approaches (including traditional and mutational breeding as well as genetic engineering) have been tried, typically with modest success (Xu et al., 2008, Plant Biotechnol J., 6:799-818 and references therein).
Although liquid biofuels offer considerable promise the reality of utilising biological material is tempered by competing uses and the quantities available. Consequently, engineering plants and microorganisms to address this is the focus of multiple research groups; in particular the accumulation of triacylglycerol (TAG) in vegetative tissues and oleaginous yeasts and bacteria (Fortman et al., 2008, Trends Biotechnol 26, 375-381; Ohlrogge et al., 2009, Science 324, 1019-1020). TAG is a neutral lipid with twice the energy density of cellulose and can be used to generate biodiesel a high energy density desirable biofuel with one of the simplest and most efficient manufacturing processes. Engineering TAG accumulation in leaves has so far resulted in a 5-20 fold increase over WT utilising a variety of strategies which includes: the over-expression of seed development transcription factors (LEC1, LEC2 and WRI1); silencing of APS (a key gene involved in starch biosynthesis); mutation of CGI-58 (a regulator of neutral lipid accumulation); and upregulation of the TAG synthesising enzyme DGAT (diacylglycerol O acyltransferase, EC 2.3.1.20) in plants and also in yeast (Andrianov et al., 2009, Plant Biotech J 8, 1-11; Mu et al., 2008, Plant Physiol 148, 1042-1054; Sanjaya et al., 2011, Plant Biotech J 9, 874-883; Santos-Mendoza, et al., 2008, Plant J 54, 608-620; James et al., 2010, Proc. Natl. Acad. Sci. USA 107, 17833-17838; Beopoulos et al., 2011, Appl Microbiol Biotechnol 90, 1193-1206; Bouvier-Nave et al., 2000, Eur J Biochem 267, 85-96; Durrett et al., 2008, Plant J 54, 593-607). However, it has been acknowledged that to achieve further increases in TAG, preventing its catabolism may be crucial within non oleaginous tissues and over a range of developmental stages (Yang and Ohlrogge, 2009, Plant Physiol 150, 1981-1989).
Positively manipulating the yield and quality of triacylglycerides (TAG) in eukaryotes is difficult to achieve. The enzyme diacylglycerol-O-acyltransferase (DGAT) has the lowest specific activity of the Kennedy pathway enzymes and is regarded as a ‘bottleneck’ in TAG synthesis.
Attempts have been made previously to improve DGAT1 by biotechnological methods, with limited success. For example Nykiforuk et al., (2002, Biochimica et Biophysica Acta 1580:95-109) reported N-terminal truncation of the Brassica napus DGAT1 but reported approximately 50% lower activity. McFie et al., (2010, JBC., 285:37377-37387) reported that N-terminal truncation of the mouse DGAT1 resulted in increased specific activity of the enzyme, but also reported a large decline in the level of protein that accumulated.
Xu et al., (2008, Plant Biotechnology Journal, 6:799-818) recently identified a consensus sequence (X-Leu-X-Lys-X-X-Ser-X-X-X-Val) (SEQ ID NO: 158) within Tropaeolum majus (garden nasturtium) DGAT1 (TmDGAT1) sequences as a targeting motif typical of members of the SNF1-related protein kinase-1 (SnRK1) with Ser being the residue for phosphorylation. The SnRK1 proteins are a class of Ser/Thr protein kinases that have been increasingly implicated in the global regulation of carbon metabolism in plants, e.g. the inactivation of sucrose phosphate synthase by phosphorylation (Halford & Hardie 1998, Plant Mol Biol. 37:735-48. Review). Xu et al., (2008, Plant Biotechnology Journal, 6:799-818) performed site-directed mutagenesis on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue (S197) in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis.
It would be beneficial to provide improved forms of DGAT1, which overcome one or more of the deficiencies in the prior art, and which can be used to increase cellular oil production.
It is an object of the invention to provide enhanced DGAT1 proteins and methods for their use to alter at least one of cellular lipid production and cellular lipid profile, and/or at least to provide the public with a useful choice.